Immunoassay techniques have been known for the last few decades and are now commonly used in medicine for a wide variety of diagnostic purposes to detect target analytes in a biological sample. Immunoassays exploit the highly specific binding of an antibody to its corresponding antigen, wherein the antigen is the target analyte. Typically, quantification of either the antibody or antigen is achieved through some form of labeling such as radio- or fluorescence-labeling. Sandwich immunoassays involve binding the target analyte in the sample to the antibody site (which is frequently bound to a solid support), binding labeled antibody to the captured analyte, and then measuring the amount of bound labeled antibody, wherein the label generates a signal proportional to the concentration of the target analyte inasmuch as labeled antibody does not bind unless the analyte is present in the sample.
A problem with this general approach is that many patients have circulating endogenous antibodies, or “autoantibodies” against an analyte of clinical interest. For example, autoantibodies have been described for cardiac troponin, myeloperoxidase (MPO), prostate specific antigen (PSA), and thyroid stimulating hormone (TSH), and other clinically significant analytes. Autoantibodies create interference in typical sandwich immunoassays that are composed of two or more analyte-specific antibodies. For example, cardiac troponin-reactive autoantibodies may interfere with the measurement of cTnI using conventional midfragment-specific immunoassays. Thus, interference from autoantibodies can produce erroneous results, particularly near the cut-off values established for clinical diagnoses, and increases the risk of false negative diagnostic results and the risk that individuals will not obtain a timely diagnosis.
One approach to addressing this problem is to choose analyte-specific antibodies that bind to specific epitopes distinct from the analyte epitopes that react with the autoantibodies. Following this general approach, efforts have focused on exploring the use of thousands of different combinations of two, three and even four analyte-specific antibodies to avoid interference from autoantibodies. However, this effort has been largely unsuccessful. It is now evident that autoantibodies against complex protein analytes are likely to be polyclonal within a particular sample, and may be even more diverse among samples from different individuals. Interference from diverse polyclonal autoantibodies may explain the observation that as little as 25% or even less of an analyte protein sequence binds to analyte-specific antibodies, which may in turn explain the lack of success using this approach.
A need exists in the art for new immunoassay methods that compensate for interference by autoantibodies in a sample, and in particular for such methods that do so without involving redesign of the analyte detection or capture antibodies.